Angelamaria Rizzo

Aryl hydrocarbon receptor-induced signals up-regulate IL-22 production and inhibit inflammation in the gastrointestinal tract.

BACKGROUND & AIMS The pathogenesis of inflammatory bowel disease (IBD) is believed to involve an altered balance between effector and regulatory T cells. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor that mediates the toxicity of dioxins, controls T-cell responses. We investigated the role of AhR in inflammation and pathogenesis of IBD in humans and mouse models. METHODS AhR expression was evaluated in intestinal tissue samples from patients with IBD and controls by real-time polymerase chain reaction (PCR) and flow cytometry. Intestinal lamina propria mononuclear cells (LPMCs) were activated in the presence or absence of the AhR agonist 6-formylindolo(3, 2-b)carbazole (Ficz). Colitis was induced in mice using trinitrobenzene sulfonic acid (TNBS), dextran sulfate sodium (DSS), or T-cell transfer. Mice were given injections of Ficz or the AhR antagonist 2-metyl-2H-pyrazole-3-carboxylic acid; some mice first received injections of a blocking antibody against interleukin (IL)-22. Cytokines were quantified by real-time PCR and flow cytometry. RESULTS Intestine tissue from patients with IBD expressed significantly less AhR than controls. In LPMCs from patients with IBD, incubation with Ficz reduced levels of interferon gamma (IFN)-γ and up-regulated IL-22. Mice injected with Ficz were protected against TNBS-, DSS-, and T-cell transfer-induced colitis; they had marked down-regulation of inflammatory cytokines and induction of IL-22. Mice given AhR antagonist produced more inflammatory cytokines and less IL-22 and developed a severe colitis. Neutralization of endogenous IL-22 disrupted the protective effect of Ficz on TNBS-induced colitis. CONCLUSIONS AhR is down-regulated in intestinal tissue of patients with IBD; AhR signaling, via IL-22, inhibits inflammation and colitis in the gastrointestinal tract of mice. AhR-related compounds might be developed to treat patients with IBDs.

Regulation of Gut Inflammation and Th17 Cell Response by Interleukin-21

BACKGROUND & AIMS Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation. METHODS Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes. CONCLUSIONS These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

Smad7 Controls Resistance of Colitogenic T Cells to Regulatory T Cell-Mediated Suppression

BACKGROUND & AIMS Foxp3-expressing regulatory T cells (Tregs) play a key role in the maintenance of the gut immune homeostasis, and an intact transforming growth factor (TGF)-beta signaling is required for their function. In inflammatory bowel disease (IBD), the TGF-beta signaling is impaired because of high expression of the inhibitory molecule Smad7. Although no intrinsic defects in Tregs function have been shown in IBD, it is still unknown whether colitogenic T cells are susceptible to Treg-mediated suppression. In this study, we have investigated whether IBD mucosal CD4+ T cells are resistant to Tregs and whether Smad7 is involved in this process. METHODS IBD lamina propria mononuclear cells (LPMC) were cultured with or without Tregs, and proliferation was assessed by flow cytometry. Proliferation of IBD LPMC was also evaluated after Smad7 antisense oligonuclotide treatment. Treg-mediated suppression of T-cell proliferation and proinflammatory cytokine expression was investigated in murine Smad7 transgenic cells. In vivo, the Smad7-dependent resistance of colitogenic naïve T cells to Tregs was studied in the adoptive transfer model of colitis. RESULTS IBD LPMC were resistant to Treg-mediated suppression, and this phenomenon was reverted by Smad7 antisense treatment. Consistently, CD4+ T cells isolated from Smad7 transgenic mice showed high proliferation, produced considerable amount of inflammatory cytokines following activation, and induced a severe colitis when transferred in immunodeficient RAG1 knockout mice even in the presence of wild-type Tregs. CONCLUSIONS Smad7 makes CD4+ T cells resistant to Tregs-mediated suppression thus fine-tuning their proinflammatory potential.

IL‐23‐mediated regulation of IL‐17 production in Helicobacter pylori‐infected gastric mucosa

Helicobacter pylori (Hp) infection is associated with a marked infiltration of the gastric mucosa by inflammatory cells. The molecular pathways that control Hp‐associated inflammatory reaction are complex, but locally induced cytokines seem to contribute to maintaining the ongoing inflammation. We have previously shown that IL‐17 is over‐produced in Hp‐infected gastric mucosa, and that IL‐17 stimulates the synthesis of IL‐8, the major neutrophil chemoattractant. Factors/mechanisms that regulate IL‐17 expression remain, however, unknown. In this study, we initially expanded our previous data, showing that CD4+ and CD8+ T cells are a source of IL‐17 in Hp‐infected samples. Since IL‐23 enhances T cell‐derived IL‐17 during bacterial infections, we then assessed the role of IL‐23 in controlling IL‐17 expression in Hp‐colonized stomach. Using real‐time PCR and ELISA, IL‐23 was detected in all gastric biopsies, but its expression was more pronounced in Hp‐infected samples in comparison to controls. Treatment of normal gastric lamina propria mononuclear cells (LPMC) with IL‐23 enhanced Stat3 activation and IL‐17 secretion, and pharmacological inhibition of Stat3 prevented IL‐23‐driven IL‐17 synthesis. Consistently, blockade of IL‐23 in cultures of LPMC from Hp‐infected patients reduced Stat3 activation and IL‐17 production. Data show that IL‐23 is overexpressed in Hp‐infected gastric mucosa where it could contribute to sustaining IL‐17 production.

Involvement of interleukin-21 in the regulation of colitis-associated colon cancer

IL-21 expression is increased in the gut of patients with colitis-associated colon cancer, and genetic ablation or antibody neutralization of IL-21 reduces tumor size and inflammation in mice treated with dextran sulfate sodium and azoxymethane.

Interleukin-25 Inhibits Interleukin-12 Production and Th1 Cell-Driven Inflammation in the Gut

BACKGROUND & AIMS During the pathogenesis of Crohns disease (CD), interleukin (IL)-12, a cytokine produced by mucosal CD14+ monocyte-like cells, promotes tissue-damaging T helper cell (Th) 1-mediated inflammation through mechanisms that are not fully understood. IL-25 promotes Th2 cell responses by activating major histocompatibility complex class II-positive non-T and non-B cells. Because Th1 and Th2 cells, and the cytokines they release, are often mutually antagonistic, we examined whether IL-25 affects IL-12 production or Th1 cell-mediated inflammation in the gut. METHODS Studies were performed using colonic samples from patients and mice with peptidoglycan (PGN)-, 2,4,6-trinitrobenzenesulphonic acid (TNBS)-, or oxazolone-induced colitis. IL-25 receptor (IL-25R) levels were evaluated in intestinal lamina propria mononuclear cells by flow cytometry, and IL-25 levels were measured by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. Mucosal CD14+ cells from patients with CD were incubated with IL-25 and/or lipopolysaccharide or PGN. Mice were injected with IL-25, and some mice first received injections of an IL-13 blocking antibody. Cytokines were quantified by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS CD14+ cells from the mucosa of CD patients expressed IL-25R and responded to IL-25 by decreasing the synthesis of IL-12 and IL-23. IL-25 prevented PGN-induced colitis in mice. IL-25 induced IL-13 production in the colon, but IL-13 was not required for suppression of PGN colitis. IL-25 ameliorated TNBS- and oxazolone-colitis. Patients with CD or ulcerative colitis produced significantly less IL-25 compared with controls. CONCLUSIONS IL-25 inhibits CD14+ cell-derived cytokines and experimental colitis. IL-25 could be a useful treatment of CD and ulcerative colitis.

Intestinal inflammation and colorectal cancer: a double-edged sword?

Chronic inflammation is thought to be the leading cause of many human cancers including colorectal cancer (CRC). Accordingly, epidemiologic and clinical studies indicate that patients affected by ulcerative colitis and Crohns disease, the two major forms of inflammatory bowel disease, have an increased risk of developing CRC. In recent years, the role of immune cells and their products have been shown to be pivotal in initiation and progression of colitis-associated CRC. On the other hand, activation of the immune system has been shown to cause dysplastic cell elimination and cancer suppression in other settings. Clinical and experimental data herein reviewed, while confirming chronic inflammation as a risk factor for colon carcinogenesis, do not completely rule out the possibility that under certain conditions the chronic activation of the mucosal immune system might protect from colonic dysplasia.

Smad7 expression in T cells prevents colitis-associated cancer

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colorectal cancer due to chronic inflammation. In IBD, chronic inflammation relies upon a TGFβ signaling blockade, but its precise mechanistic relationship to colitis-associated colorectal cancer (CAC) remains unclear. In this study, we investigated the role of the TGFβ signaling inhibitor Smad7 in CAC pathogenesis. In human colonic specimens, Smad7 was downregulated in CD4(+) T cells located in the lamina propria of patients with complicated IBD compared with uncomplicated IBD. Therefore, we assessed CAC susceptibility in a transgenic mouse model where Smad7 was overexpressed specifically in T cells. In this model, Smad7 overexpression increased colitis severity, but the mice nevertheless developed fewer tumors than nontransgenic mice. Protection was associated with increased expression of IFNγ and increased accumulation of cytotoxic CD8(+) and natural killer T cells in the tumors and peritumoral areas. Moreover, genetic deficiency in IFNγ abolished the Smad7-dependent protection against CAC. Taken together, our findings defined a novel and unexpected role for Smad7 in promoting a heightened inflammatory response that protects against CAC.

Inhibition of monocyte-derived inflammatory cytokines by IL-25 occurs via p38 Map kinase–dependent induction of Socs-3

IL-25, a member of the IL-17 cytokine family, is known to enhance Th2-like responses associated with increased serum levels of IgE, IgG1, IgA, blood eosinophilia, and eosinophilic infiltrates in various tissues. However, IL-25 also abrogates inflammatory responses driven by Th17 cells. However, the cell types that respond to IL-25 and the mechanisms by which IL-25 differentially regulates immune reactions are not well explored. To identify potential targets of IL-25, we initially examined IL-25 receptor (IL-25R) in human peripheral blood cells. IL-25R was predominantly expressed by CD14(+) cells. We next assessed the functional role of IL-25 in modulating the response of CD14(+) cells to various inflammatory signals. CD14(+) cells responded to IL-25 by down-regulating the synthesis of inflammatory cytokines induced by toll-like receptor (TLR) ligands and inflammatory cytokines. Inhibition of cytokine response by IL-25 occurred via a p38 Map kinase-driven Socs-3-dependent mechanism. In vivo, IL-25 inhibited monocyte-derived cytokines and protected against LPS-induced lethal endotoxemia in mice. These data indicate that IL-25 is a negative regulator of monocyte proinflammatory cytokine responses, which may have therapeutic implications.

Inhibition of colitis by IL‐25 associates with induction of alternatively activated macrophages

Background: Interleukin (IL)‐25, a Th2‐related factor, inhibits the synthesis of inflammatory cytokines by macrophages and attenuates experimental colitis in mice. The mechanism underlying the counterregulatory effect of IL‐25, however, remains unknown. Since Th2‐cytokines can abrogate inflammatory pathways by inducing alternatively activated macrophages (AAMs), we evaluated whether AAMs are involved in the IL‐25‐mediated anticolitic effect. Methods: AAM‐related markers were evaluated in peritoneal and lamina propria mononuclear cells of mice with or without 2,4,6‐trinitrobenzenesulphonic acid (TNBS)‐induced colitis treated with IL‐25 and/or neutralizing IL‐4, IL‐13, and transforming growth factor beta 1 (TGF‐&bgr;1) antibodies. Peritoneal AAMs induced in vivo by injecting mice with IL‐25 were transferred to mice with TNBS colitis. Finally, we assessed the in vitro effect of IL‐25 on the alternative activation of peritoneal F4/80+ cells. Results: IL‐25 enhanced the expression of AAM‐related markers in F4/80+ cells infiltrating the peritoneum and colon of naïve and colitic mice. Peritoneal F4/80+ cells isolated from IL‐25‐treated mice reduced the severity of TNBS colitis when injected intraperitoneally to recipient mice. Since IL‐25 did not directly induce AAM in vitro and in vivo in mice, IL‐25 administration enhanced the expression of IL‐4, IL‐13, and TGF‐&bgr;1, which are known to promote AAM differentiation, we finally assessed whether such cytokines were involved in the IL‐25‐driven AAM induction. Blockade of IL‐4, IL‐13, and TGF‐&bgr;1 with neutralizing antibodies in mice did not inhibit the stimulatory effect of IL‐25 on AAM gene expression. Conclusions: The IL‐25‐mediated anticolitic effect is associated with induction of AAMs, a subset of macrophages with antiinflammatory properties. (Inflamm Bowel Dis 2012;)